The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.

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Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications

Breast cancer patients with the same stage of disease can have markedly different treatment responses and overall outcome. The strongest predictors for metastases (for example, lymph node status and histological grade) fail to classify accurately breast tumours according to their clinical behaviour. Chemotherapy or hormonal therapy reduces the risk of distant metastases by approximately one-third; however, 70-80% of patients receiving this treatment would have survived without it. None of the signatures of breast cancer gene expression reported to date allow for patient-tailored therapy strategies. Here we used DNA microarray analysis on primary breast tumours of 117 young patients, and applied supervised classification to identify a gene expression signature strongly predictive of a short interval to distant metastases ('poor prognosis' signature) in patients without tumour cells in local lymph nodes at diagnosis (lymph node negative). In addition, we established a signature that identifies tumours of BRCA1 carriers. The poor prognosis signature consists of genes regulating cell cycle, invasion, metastasis and angiogenesis. This gene expression profile will outperform all currently used clinical parameters in predicting disease outcome. Our findings provide a strategy to select patients who would benefit from adjuvant therapy.

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Gene expression profiling predicts clinical outcome of breast cancer

Background A more accurate means of prognostication in breast cancer will improve the selection of patients for adjuvant systemic therapy. Methods Using microarray analysis to evaluate our previously established 70-gene prognosis profile, we classified a series of 295 consecutive patients with primary breast carcinomas as having a gene-expression signature associated with either a poor prognosis or a good prognosis. All patients had stage I or II breast cancer and were younger than 53 years old; 151 had lymph-node–negative disease, and 144 had lymph-node–positive disease. We evaluated the predictive power of the prognosis profile using univariable and multivariable statistical analyses. Results Among the 295 patients, 180 had a poor-prognosis signature and 115 had a good-prognosis signature, and the mean (±SE) overall 10-year survival rates were 54.6±4.4 percent and 94.5±2.6 percent, respectively. At 10 years, the probability of remaining free of distant metastases was 50.6±4.5 percent in the group with a...

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A Gene-Expression Signature as a Predictor of Survival in Breast Cancer

background The likelihood of distant recurrence in patients with breast cancer who have no involved lymph nodes and estrogen-receptor–positive tumors is poorly defined by clinical and histopathological measures. methods We tested whether the results of a reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay of 21 prospectively selected genes in paraffin-embedded tumor tissue would correlate with the likelihood of distant recurrence in patients with node-negative, tamoxifen-treated breast cancer who were enrolled in the National Surgical Adjuvant Breast and Bowel Project clinical trial B-14. The levels of expression of 16 cancerrelated genes and 5 reference genes were used in a prospectively defined algorithm to calculate a recurrence score and to determine a risk group (low, intermediate, or high) for each patient. results Adequate RT-PCR profiles were obtained in 668 of 675 tumor blocks. The proportions of patients categorized as having a low, intermediate, or high risk by the RT-PCR assay were 51, 22, and 27 percent, respectively. The Kaplan–Meier estimates of the rates of distant recurrence at 10 years in the low-risk, intermediate-risk, and high-risk groups were 6.8 percent (95 percent confidence interval, 4.0 to 9.6), 14.3 percent (95 percent confidence interval, 8.3 to 20.3), and 30.5 percent (95 percent confidence interval, 23.6 to 37.4). The rate in the low-risk group was significantly lower than that in the high-risk group (P<0.001). In a multivariate Cox model, the recurrence score provided significant predictive power that was independent of age and tumor size (P<0.001). The recurrence score was also predictive of overall survival (P<0.001) and could be used as a continuous function to predict distant recurrence in individual patients. conclusions The recurrence score has been validated as quantifying the likelihood of distant recurrence in tamoxifen-treated patients with node-negative, estrogen-receptor–positive breast cancer.

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A Multigene Assay to Predict Recurrence of Tamoxifen-Treated, Node-Negative Breast Cancer

Morphological assessment of the degree of differentiation has been shown in numerous studies to provide useful prognostic information in breast cancer, but until recently histological grading has not been accepted as a routine procedure, mainly because of perceived problems with reproducibility and consistency. In the Nottingham/Tenovus Primary Breast Cancer Study the most commonly used method, described by Bloom & Richardson, has been modified in order to make the criteria more objective. The revised technique involves semiquantitative evaluation of three morphological features--the percentage of tubule formation, the degree of nuclear pleomorphism and an accurate mitotic count using a defined field area. A numerical scoring system is used and the overall grade is derived from a summation of individual scores for the three variables: three grades of differentiation are used. Since 1973, over 2200 patients with primary operable breast cancer have been entered into a study of multiple prognostic factors. Histological grade, assessed in 1831 patients, shows a very strong correlation with prognosis; patients with grade I tumours have a significantly better survival than those with grade II and III tumours (P less than 0.0001). These results demonstrate that this method for histological grading provides important prognostic information and, if the grading protocol is followed consistently, reproducible results can be obtained. Histological grade forms part of the multifactorial Nottingham prognostic index, together with tumour size and lymph node stage, which is used to stratify individual patients for appropriate therapy.

Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up.

This study was carried out in an effort to resolve the dilemma created by three proposed modifications of the Dukes' C class of colorectal cancer. Each modification is based on a separate prognostic discriminant characterized by: the level of histologically positive nodes, the depth of tumor penetration, and the number of histologically positive nodes. Data were derived from 844 patients with Dukes' C lesions randomized into two prospective clinical trials of the NSABP; the mean time on study was 41 months. Analysis of the three modifications as independent variables without regard for possible confounding effects disclosed that each had a highly significant predictive capacity. When each discriminant was examined, this time adjusting for the contribution of the other two discriminants, the effect attributable to the level of positive nodes was markedly attenuated. Thus, the level of positive nodes provided little information over and above that of depth of tumor penetration and the number of positive nodes. Of the two latter discriminants, although both were significant predictors of survival, the number of positive nodes appeared to be the strongest factor. Using both depth of penetration and the number of positive nodes, a unique Dukes' C subset of patients could be identified with a prognosis at least as good as Dukes' B lesions; this group was characterized by partial tumor penetration and the presence of 1-4 positive nodes. It is concluded that both depth of penetration and the number of positive nodes represent appropriate modifications of the initial Dukes scheme, and one discriminant should not be used to the exclusion of the other. The data raise serious doubts relative to the propriety of newly proposed TNM classification schemes that fail to utilize the number of positive nodes as a predictive discriminant.

The prognostic value of the modifications of the Dukes' C class of colorectal cancer. An analysis of the NSABP clinical trials.

Two‐hundred seventy eight of 565 patients treated by radical mastectomy for invasive breast cancer in a prospective, randomized clinical trial exhibited axillary nodal metastases. Patients in whom the largest nodal metastasis measured ≤2 mm in its greatest diameter were regarded as having micrometastases and those in whom the lesions were ≤2 mm as having macrometastases. Contingency table analysis disclosed that the group with macrometastases had a greater likelihood that they would be clinically assessed as stage II, their primary tumors would be ≤2 cm, they would have 4 or more positive nodes, extension of the metastasis through the nodal capsule would have occurred and uninvolved nodes would exhibit a germinal center predominance pattern. Life table analyses failed to reveal any significant difference in survival between patients with micrometastases and those without nodal metastases although both of these groups exhibited significantly greater survival than patients with macrometastases. On the other hand, treatment failure rates were more comparable in patients with micrometastases to those with macrometastases rather than absent nodal involvement. Multivariate analysis revealed that these apparent effects on survival and treatment failure rates in patients with micro and macrometastases were more directly related to the number of nodes involved with metastases rather than the size of the latter. A subset of patients with micrometastases in whom the metastases measured ≥1.3 mm was identified who exhibited survival and treatment failure rates similar to those of the negative node patients. The data, although based on a relatively small sample size, suggest metastases ≥1.3 mm may exert an influence independent of number of nodes involved. The excellent prognosis of patients with these occult nodal metastases (≥1.3 mm) warrants the designation of the stage of their disease as 1 1/2. Cancer 42:2032–2038, 1978.

Pathologic findings from the National Surgical Adjuvant Breast Project (Protocol No. 4). V. Significance of axillary nodal micro- and macrometastases.

Histologic grading of breast cancer.

Association of tamoxifen and uterine sarcoma.

In 1972, a prospective, randomized, multi-institutional, cooperative clinical trial was begun to evaluate the efficacy of prolonged 1-phenylalanine mustard (L-PAM) administration following operation in lengthening the disease free interval of patients with primary breast cancer That protocol using a single agent was the first of a series directed toward evaluating successively more complex chemotherapeutic regimens in an attempt to define subsets of patients which might be responsive to less therapy than others When it was observed that L-PAM prolonged the disease free interval, particularly of premenopausal patients, findings were reported and a new evaluation comparing L-PAM with L-PAM plus 5-fluorouracil (5-FU) was begun Upon completion of patient accrual in that protocol, an additional trial comparing L-PAM and 5-FU with L-PAM, 5-FU and Methotrexate was implemented The present report updates findings from the initial study and presents those from the second It compares results across the first two protocols as well as between groups within a protocol While insufficient time has elapsed for determining the ultimate worth of the modalities employed, findings from the second protocol confirm those previously reported indicating that L-PAM lengthens the disease free interval following mastectomy The combination of L-PAM with 5-FU resulted in a reduction of treatment failure at 12 months which is as good or better than that observed with L-PAM in the first protocol lending further credibility to the earlier findings While at the end of the first year following mastectomy there was alomst a 50% reduction in treatment failures in patients aged 50 or over (post-menopausal), by 18 months the reduction was 23% and at two years, based on small numbers of patients, only 5% Examination of results from the first protocol (placebo vs L-PAM) after two years reveals a most highly significant effect of L-PAM in pre-menopausal women with one to three positive nodes There is an 89% reduction of treatment failures A similar but less striking effect is noted for those under 50 with ≥four positive nodes In older patients in both nodal categories, the early observed effect for L-PAM has decreased with time Inter-protocol comparisons relative to survival are premature At two years survival in L-PAM patients is 36% greater than in those receiving placebo It is somewhat better in every subgroup for those receiving L-PAM Information relative to the effect of these agents on patient toxicity and loco-regional treatment failures is presented All of the findings stress the urgency for obtaining results on subsets of patients rather than on a population as a whole and they lend support to the thesis that since breast cancer is an eponym to describe a heterogeneous group of tumors residing in a heterogeneous group of women, it is unlikely that uniformly qualitative and quantitative systemic regimens of therapy will be required for every patient

L-phenylalanine mustard (L-PAM) in the management of primary breast cancer: An update of earlier findings and a comparison with those utilizing L-PAM plus 5-fluorouracil (5-FU)

Bernard Fisher

Papers

The prognostic value of the modifications of the Dukes' C class of colorectal cancer. An analysis of the NSABP clinical trials.

Pathologic findings from the National Surgical Adjuvant Breast Project (Protocol No. 4). V. Significance of axillary nodal micro- and macrometastases.

Histologic grading of breast cancer.

Association of tamoxifen and uterine sarcoma.

L-phenylalanine mustard (L-PAM) in the management of primary breast cancer: An update of earlier findings and a comparison with those utilizing L-PAM plus 5-fluorouracil (5-FU)